Molecular physiology of phosphoryl group transfer from carbamoyl phosphate by a hyperthermophilic enzyme at low temperature

Biochemistry
Santiago Ramón-MaiquesVicente Rubio

Abstract

Enzymes from thermophilic organisms often exhibit low activity at reduced temperature. To obtain a better understanding of this sluggishness, we have studied the reaction at 24 degrees C of the carbamate kinase (CK) from the hyperthermophile Pyrococcus furiosus. This enzyme is much slower at low temperature than is the CK from the mesophile Enterococcus faecalis. X-ray structures demonstrated bound ADP (even when no nucleotide was added) with the hyperthermophilic but not with the mesophilic CK. We use centrifugal gel filtration, rate of dialysis and pulse-chase experiments to demonstrate that the pyrococcal enzyme, at 24 degrees C, binds ADP avidly (K(D) = 34 nM), that ADP dissociates from this complex with a t1/2 value of 2.4 s, and that ADP binding is very fast (kappa = 8.4 x 10(6) M(-1) x s(-1)). The high affinity, rather than restrictions to dissociation, explains the isolation of the pyrococcal enzyme as an ADP complex. Carbamoyl phosphate adds quickly to this complex, and ADP cannot dissociate from the resulting ternary complex, being that it is converted very slowly (t1/2 = 10.3 s) to ATP, which dissociates quickly (t1/2 < 2.4 s). The slow conversion is a part of the normal enzyme reaction and limits the rate of the rea...Continue Reading

Citations

Jun 19, 2013·Proceedings of the National Academy of Sciences of the United States of America·Naoko MizunoSantiago Ramón-Maiques

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